ORIGINAL ARTICLE Atrazine degradation by stable mixed cultures enriched from agricultural soil and their characterization S. Siripattanakul 1,2 , W. Wirojanagud 3 , J. McEvoy 4 , T. Limpiyakorn 5 and E. Khan 2 1 National Center of Excellence for Environmental and Hazardous Waste Management, Chulalongkorn University, Bangkok, Thailand 2 Department of Civil Engineering, North Dakota State University, Fargo, ND, USA 3 Department of Environmental Engineering and Research Center for Environmental and Hazardous Substance Management, Khon Kaen University, Khon Kaen, Thailand 4 Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND, USA 5 Department of Environmental Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand Introduction Atrazine (6-chloro-N-ethyl-N-(1-methylethyl)-1,3,5-tri- azine-2,4-diamine), one of the most widely used herbi- cides, has been applied to control broad-leaf weeds for corn, sorghum, sugarcane and other crops. The wide- spread use of this herbicide has led to its contamination in different environmental media (Wauchope 1978; Jaya- chandran et al. 1994; Koskinen and Clay 1997). Atrazine concentrations above the allowable contaminant levels for drinking water of 0Æ1 and 3 lgl )1 in Europe and the United States, respectively, have been frequently detected (Rousseaux et al. 2003). Biodegradation is one of the key attenuation processes of atrazine in the environment. Previous research on atra- zine biodegradation focused on pure atrazine-degrading cultures (Mandelbaum et al. 1995; Radosevich et al. 1995; Struthers et al. 1998; Strong et al. 2002; Piutti et al. 2003; Rousseaux et al. 2003; Aislabie et al. 2005). Several atrazine-degrading micro-organisms were isolated from atrazine spilled sites or atrazine applied crop fields includ- ing Pseudomonas, Rhizobium and Agrobacterium strains. Among atrazine-degrading species, two main biodegrada- tion pathways via hydrolytic dechlorination and N-deal- kylation convert atrazine to hydoxyatrazine (HA) and desethylatrazine (DEA) or deisopropylatrazine (DIA), Keywords Agrobacterium, atrazine biodegradation, identification, Klebsiella, mixed cultures. Correspondence Eakalak Khan, Department of Civil Engineering, North Dakota State University, 1410 14th Avenue North, Civil and Industrial Engineering Building (Room 201), Fargo, ND 58105, USA. E-mail: eakalak.khan@ndsu.edu 2008 ⁄ 0486: received 20 March 2008, revised 16 August 2008 and accepted 10 September 2008 doi:10.1111/j.1365-2672.2008.04075.x Abstract Aims: The aim of this work was to enrich stable mixed cultures from atrazine- contaminated soil. The cultures were examined for their atrazine biodegrada- tion efficiencies in comparison with J14a, a known atrazine-degrading strain of Agrobacterium radiobacter. The cultures were also characterized to identify community structure and bacterial species present. Methods and Results: The cultures were enriched and then stabilized in bacte- rial media. The stable mixed cultures and J14a were tested in a medium con- taining 100 lgl )1 of atrazine. For all cultures, atrazine was removed 33–51% within 7 days and the cell optical density increased from 0Æ05 to between 0Æ50 and 0Æ70. Four isolates designated ND1, ND2, ND3 and ND4 were purified from the mixed cultures and identified based on sequence analysis of the 16 S rRNA gene as Alcaligenes faecalis, Klebsiella ornithinolytica, Bacillus megaterium and Agrobacterium tumefaciens, respectively. An atrazine-degrading gene, atzA, was present in ND2 and ND4. Conclusions: The stable mixed cultures obtained could degrade atrazine. Klebsiella ornithinolytica ND2 and Ag. tumefaciens ND4 are atrazine degraders. Significance and Impact of the Study: The novel stable mixed cultures could be used for bioremediating crop fields contaminated with atrazine. This is the first report of the atzA gene in Kl. ornithinolytica. Journal of Applied Microbiology ISSN 1364-5072 986 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 986–992 ª 2009 The Authors